Evaluation of TNF-α cytokine production in patients with tuberculosis compared to healthy people

Background: Mycobacterium tuberculosis (TB) is one of the most important causes of human mortality. Approximately one-third of the world’s population is infected with TB and 5–10% of them develop the active form of the disease. Cytokines play a major role in the host defense process against Mycobacterium infections. Among these cytokines, tumor necrosis factor alpha (TNF-α) has a prominent role in the defense of and pathological responses to tuberculosis. Materials and methods: A case-control study was carried out from May 2016 to June 2017. 45 patients with diagnosis of tuberculosis (smear and positive culture) were included as case group and 45 healthy subjects as control group. The serum levels of TNF-α were determined with the enzyme-linked immunosorbent assay (ELISA) method. Results: The concentration of TNF-α in patients with TB was significantly higher than in the control group (P<0.05). However, the difference was only significant in the age groups 20–30 and 50–60 years; in the age groups 30–40, 40–50 and 50–70 years, the difference was not significant, although certain trends were apparent. Discussion and conclusion: Since the level of serum TNF-α is higher in patients with pulmonary tuberculosis than in individuals without it, the measurement of TNF-α levels can be useful as a probable marker for the diagnosis of tuberculosis.


Introduction
Mycobacterium tuberculosis (TB) is one of the most important causes of mortality in humans. According to the World Health Organization report in 2017, tuberculosis is responsible for 1.7 million deaths every year [1]. About one-third of the world population is infected, but only about 10% of these develop active tuberculosis [2]. The causative agent is the intracellular bacterial pathogen itself; its antigens have the ability to stimulate the production of cytokines via the mononuclear phagocyte system. Studies show that susceptibility to the disease varies among individuals, e.g., not everyone who is exposed to this bacterium becomes infected with TB [3]. The course of the disease also differs from person to person. These differences can be due to host factors and the genetic sensitivity of various individuals [3]. In this disease, cellular immunity and cytokines are intermediaries in the immune system and inflammatory responses [4], [5]. It seems that the acquired immune response in the pathway of T-helper lymphocytes can limit and stop bacterial growth. These cells secrete cytokines, such as interferon gamma and TNF-α, and stimulate macrophages to further produce more active oxygen and free radicals. As a result, cells are better able to kill microbes, and more microbial antigens are delivered to T cells [6], [7]. T-helper 2-type cytokines such as IL-4, IL-5, IL-10, IL-13 influence the course of the disease [8]. Tumor Necrosis Factor-α is a cytokine produced by many types of cells, such as macrophages and monocytes. TNF-α exists in two forms; a type II transmembrane protein, and a mature soluble protein.
However, this cytokine is initially produced as a membrane protein of 212 amino acids [9]. TNF-α binding to cell surface receptors TNFRp55 (TNF-R1, CD120a or p55/60) and TNF Rp75 (aR2, CD120b and p75/80) triggers an intracellular signaling cascade. The Rp55 receptor is expressed by most tissues, and TNF-α terraform solution is mainly attached to Rp55 receptor, although activated by both types of TNF-α. The membrane associated form mostly binds to TNF Rp75, which is specific to the immune system cells [10]. Awareness of the specific functions and the activation of TNF-α receptors considerably help to understand the inhibitory effects of TNF-α on some diseases [11]. The purpose of this study was to determine the importance of TNF-α in protecting against tuberculosis and investigate the rate of production of this cytokine in patients with tuberculosis as compared to the control group.

Study design
A case-control study was conducted from June 2016 to July 2017. The case group consisted of 45 patients with a definite tuberculosis diagnosis (with smear and positive culture), and 45 healthy subjects were selected as the control group.

Statistical analysis
ANOVA, Tukey's test, and Bonferroni correction were employed, as was the Shapiro-Wilk test (P≤0.05) due to the low sample size.

Results
The age of patients ranged from 20 to 60 years, with a mean age of 41±1.5 years. The study population included 52 (68.8%) men and 38 (42.2%) women. The mean serum level of TNF-α in the patient group were 10.6 pg/ml with the lowest and highest rates being 1.2 and 186.2 pg/ml, respectively. The mean serum level of TNF-α in the control group was 8.26 pg/ml, with the lowest and highest rates being 1.2 and 240 pg/ml, respectively ( Figure 1, Figure 2). Data were evaluated in Prism 6 GraphPad software, using one-way ANOVA and Tukey's tests. A comparison of serum levels of TNF-α with different age groups is shown in Figure 3. The results show that between the control group and the patient group, there was a significant difference (P<0.05) in the age groups 20-30 and 50-60 years of age. There were no significant differences in the other age groups (Table 1).

Discussion
Pulmonary tuberculosis is one of the most important infectious diseases in humans. Despite the use of antituberculosis drugs, this disease is one of the major causes of mortality, especially in developing countries. In recent years, researchers attempted to find a link between serum cytokines TNF-α, IFN-γ, IL-12, IL-10, and Mycobacterium tuberculosis. The results of this study showed that TNF-α production in patients with Mycobacterium tuberculosis infection was greater than serum levels of TNF-α in the control group. Cytokines play a crucial role in the host defense process against Mycobacterium infections [12]. Among these cytokines, TNF-α has a prominent role in defense and pathological responses to tuberculosis [13], [14]. In a study by Anoosheh et al. [12], cytokine gene polymorphisms in PTB and healthy controls were evaluated. The results demonstrated the importance of cytokine TNF-α. The findings of other authors also showed a significant correlation between patient and control groups (P<0.05) [11]. In this study, serum levels of TNF-α were significantly higher in the patient group than in the control group. Our findings suggest that serum levels of TNF-α increase in patients  The results of their study, which were similar to those of the present study, showed a significant increase in serum levels of TNF-α in the patient group compared to the control group (P<0.001). Other studies have shown the importance of TNF-α in patients with tuberculosis [16], [17], [18]. The results of these studies showed that the serum levels of TNF-α decreased significantly with treatment. Nakya et al. [19] studied serum levels of TNF-α in patients with active pulmonary tuberculosis compared to a control group. The results of that study showed that the serum level of TNF-α increased significantly in the patient group compared with the control group, which agrees 3/5 GMS Hygiene and Infection Control 2018, Vol. 13, ISSN 2196-5226 Table 1: Analysis of TNF-α serum levels by age groups with our results (P<0.001). In the study by Shameem et al. [20], serum levels of TNF-α were investigated in TB patients. The results did not demonstrate a significant relationship between the control group and the patients, which contradicts the results of this study (P<0.05). In another study [21], the serum levels of TNF-α in Mycobacterium tuberculosis and non-tuberculosis diseases were measured. The results of that study showed that the serum levels of TNF-α in patient with Mycobacterium tuberculosis did not differ significantly compared to healthy controls. This contradicts the results of the current study. In another study, the serum level of TNF-α was measured in peripheral blood monocytes of Mycobacterium and control patients [22]. The difference between results from various studies is correlated with the geographic location of the samples and the type of Mycobacteria prevalent in the studied area.

Conclusion
The findings of this study show that cytokines can be used as a possible marker for the diagnosis of tuberculosis. Measuring the level of cytokines can be used to supplement the diagnostic tests for Mycobacterium. More studies are needed to confirm these findings.

Notes
Competing interests